The rabbit cortical collecting duct is thought to play an important role in the regulation of Na+, K+, Cl- and H+ excretion. Isolated perfused tubules of this segment absorb Na+ and Cl- from the tubule lumen and secrete H+ (or absorb HCO3-) and K+ into the lumen. The rates vary depending upon the luminal and metabolic status of the animal and the experimental conditions. Morphologically, the epithelium is characterized by two cell types, the principal cell and the intercalated cell, which likely have different functions. The aim of the present proposal is to assess the transport function of these two cell types and to elucidate the underlying cellular mechanisms which are responsible for ion transport in the isolated perfused cortical collecting duct. Conventional and ion-selective microelectrode will be employed to impale individual cells to directly measure intracellular voltages and ionic activities and hence, the electrochemical gradients for each ion. These estimates will be combined with measurements of transepithelial voltages, conductances and iosotopic ionic fluxes to evaluate the distribution of fluxes between transcellular and paracellular pathways, the passive ionic permeabilities and associated ionic fluxes across individual cell membranes, the active transport steps, and the coupling of flows between ions. The mechanism of regulation of H+ secretion and its relation to a H+ ATPase (the suspected enzymatic equivalent of the H+-pump) and insertion of active pumps into the membrane will be evaluated from rates of H+ secretion, H+ ATPase activity and use of fluorescent tags to follow insertion of pump units into the membrane. This will be correlated with regulation of Na+ and K+ fluxes and Na-K-ATPase activity. The studies should provide important new insights into the intraepithelial location and cellular mechanisms of ion transport by the cortical collecting tubules. This should provide an important framework by which to evaluate and correct inappropriate alterations in electrolyte excretion observed with some altered hormonal and metabolic states, with various disease states, and with transplanted kidneys.